Turner syndrome (TS), which is caused by the complete or partial loss of the second sex chromosome, is the most commonly occurring sex chromosome aneuploidy. Individuals with TS are phenotypically female as they retain one complete copy of the X chromosome. This condition results in a greatly increased susceptibility for common diseases that have a significant sex bias. Indeed the poor health trajectory in TS resulting in early death from cardiovascular-related diseases is unprecedented. In particular, individuals with TS are ~100-times more likely than a euploid woman to be born with a bicuspid aortic valve (BAV). BAV is the most commonly occurring congenital heart defect in humans, and is most often seen in males in the euploid population. Having a BAV predisposes affected individuals to adult valve disease, including calcification, valve thickening and stenosis. Aortic regurgitation can occur promoting an increased risk for infective endocarditis. Thoracic aortic aneurysms occur in nearly 50% of people with BAV, which can result in aortic dissection and death. The relationship between having a congenital heart defect, BAV, and later onset aortic disease (known as bicuspid aortic valve disease or BAVD) is not well understood. There is evidence that altered hemodynamic flow plays a role in aneurysm progression, but it is clear that individuals with BAVD have an aneurysm susceptible aorta for unknown reasons. Despite the seriousness of this disease, few advances have been made in understanding the etiology. Searches for genetic variants that underlie the cause of BAVD have been of low yield indicating that there is genetic heterogeneity or that other mechanisms are in play. One possibility is that differences in epigenetic regulation of genes involved in aortic valve formation, and aortic wall development and homeostasis may be risk factors. It is known that the epigenetic landscape in TS differs significantly between TS and euploid individuals, and that there is differential gene expression in TS. In this pilot project we propose to explore individual differences in epigenetic regulation of gene expression using TS as a phenotypically extreme model of BAVD risk. Our preliminary data shows differential methylation of genes in the NOTCH1 pathway between TS individuals with BAVD (cases) and those with no BAVD (controls). This is significant as mutations in NOTCH1 are known to cause non-syndromic familial BAVD. In this study we will use DNA methyl-capture sequencing to identify differences in gene methylation patterns in a larger TS cohort to confirm this finding (aim 1). In aim 2 we will explore the utility of using iPS cell-derived vascular smooth muscle cell lines as a model system for differential epigenetic regulation of genes associated with BAVD. We have unique access to the TS cohort through the NHLBI GenTAC repository in BioLINCC to support this study. This project is directly responsive to the NHLBI Strategic Vision, particularly with regards to the mandate under objective 3 to study epigenetic factors that determine sex-related differences in cardiovascular disease. Data from this study, with a focus on candidate genes for BAVD, will provide insight into the role of epigenetics in this serious disease.